Project Summary/Abstract Despite the absolute dependence of the pathology and symptoms of painful pulpitis on bacteria, the mechanisms by which bacteria produce pain in the pulp are not known. In order to identify therapeutic targets to treat painful pulpitis, this project will identify the critical mechanisms mediating LPS-evoked pulpal pain. The long-term goal is to identify novel therapeutic targets to treat painful oral infections. The central hypothesis is that TLR4- TRPA1 receptor interdependent signaling is required for the initial activation of sensory neurons by LPS, while prolonged LPS-evoked pathological neuroplasticity is dependent on TLR4 signaling. This will be tested by completing the following Specific Aims: Aim1 Identify the mechanisms by which LPS initiates symptomatic pulpitis. The hypothesis that LPS causes painful pulpitis by activating sensory neurons through TLR4-initiated signaling that also requires TRPA1 channel activation will be tested. With genetic and pharmacologic approaches, the effects of interfering with TLR4 and TRPA1 receptor signaling on LPS-stimulated nocifensive behaviors and activation of trigeminal sensory neurons will be measured. Further the pathway by which TLR4 activates TRPA1 will be determined. Aim 2 Identify the mechanism by which LPS causes pathological neuroplasticity during pulpitis. The hypothesis that the LPS-mediated neuroplasticity is dependent on TLR4 signaling, and interference with this pathway is needed to reverse established pathological changes in the nervous system. With genetic and pharmacologic approaches the effects of interfering with TLR4 and TRPA1 receptor signaling on LPS-stimulated pathological changes in the trigeminal nucleus (cytokine/chemokine expression, glial upregulation, Fos expression) will be measured. Aim 3 Determine whether neuroinflammatory effects of LPS in human dental pulp are TLR4 or TRPA1 dependent. The hypothesis is that both TRPA1 and TLR4 receptors are required for LPS-evoked CGRP secretion while TLR4 primarily mediates cytokine secretion from human pulp. Using an ex vivo model of human pulp slices the effects of pharmacologic inhibition of TLR4 and TRPA1 on LPS stimulated neurosecretion and cytokine release from pulp will be measured. Upon completion the receptors and signaling pathways mediating LPS evoked pulpal pain will be determined. The scientific contribution provided by these studies is significant because it will identify bacterial specific pain mechanisms, allowing for identification of targets to alleviate symptoms of painful pulpitis while maximally conserving pulp and dental hard tissues. The identification of bacterial-specific pain mechanisms relevant to pulpitis and other painful infections will fundamentally improve health by providing the rationale to pursue precise analgesic therapies.